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The Jackson Laboratory, Bar Harbor, ME 04609
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells mediated by both CD4 and CD8 T
cell responses (reviewed in Refs. 1 and 2).
Multiple susceptibility (Idd) genes contribute to IDDM
development (reviewed in Refs. 3 and 4).
However, while polygenically controlled, particular MHC haplotypes
provide the primary genetic component of IDDM susceptibility in both
humans and NOD mice (reviewed in Refs. 4 and
5). Within the MHC, specific combinations of HLA-DQ and
-DR class II alleles provide a large component of IDDM susceptibility
in humans by mediating cell autoreactive CD4 T cell responses
(5). Similarly, IDDM development in NOD mice requires that
the rare H2-Ag7 MHC class II gene product
(homologue of human DQ8) be homozygously expressed. This was
demonstrated by the fact that transgenes encoding H2-A variants from
diabetes-resistant MHC haplotypes confer dominant IDDM resistance to
NOD mice (6, 7, 8, 9, 10). However, NOD mice also normally fail to
express an H2-E MHC class II variant (homologue of human DR) due to a
deletion in the first exon of the gene encoding its 
-chain subunit
(11). The lack of H2-E expression also contributes to IDDM
susceptibility in NOD mice, because reversal of this defect with
Ea transgenes inhibits the development of disease (7, 12, 13). Certain human MHC class II gene products, such as DQ6,
also appear to confer dominant IDDM resistance (5).
Because DR products are always expressed on human APC, it is unclear
whether specific alleles in linkage disequilibrium with diabetogenic DQ
alleles actually contribute to increased disease risk, are neutral
(conferring no protection), or confer different degrees of protection
(14).
MHC class II expression is largely restricted to hemopoietically
derived APC, which include B lymphocytes, macrophages, and DC (reviewed
in Ref. 15). Hence, it is not surprising that APC defects
controlled by the unusual class II variants of the
H2g7 MHC haplotype, but also partially
dependent on contributions from other Idd genes, are largely
responsible for IDDM development in NOD mice (reviewed in Ref.
2). These defects entail a reduced ability of NOD APC to
mediate tolerogenic functions that would normally block the development
of autoreactive T cells. APC are also essential to the subsequent
functional activation of the pancreatic cell autoreactive CD4 T
cells that are generated as a result of these tolerance induction
defects in NOD mice. B lymphocytes have been shown to be the most
important subpopulation of APC in NOD mice for activating the effector
activity of cell autoreactive CD4 T cells (16, 17, 18, 19).
Collectively, these previous findings indicate that APC could be an
ideal target for gene therapy protocols that may inhibit IDDM
development. The transgenic studies described above indicate that an
effective IDDM prevention protocol might be one that causes APC to
express a dominantly protective MHC class II gene product. However, a
prerequisite to the success of such protocols is to determine what
particular subtype(s) and proportions of APC must express a particular
MHC class II variant to inhibit IDDM development. Furthermore, the
ability to use APC expressing potentially protective MHC class II
variants to block IDDM development might also be enhanced by
determining their disease inhibitory mechanisms.
In the current study we evaluated what subtypes and proportions of APC
must express transgenically restored H2-E MHC class II molecules to
inhibit IDDM development in NOD mice and tested a previously proposed
mechanism of protection. The issue of what APC subtypes exert
H2-E-mediated IDDM protective effects was addressed through the use of
Ea transgenic NOD mice that were made deficient in B
lymphocytes by introduction of a functionally inactivated
Igµ gene (designated
Igµnull). We had previously reported
that a possible mechanism by which IDDM development is inhibited by
transgenic H2-E expression in NOD mice might entail an alteration in
the pattern of cytokines produced by cell autoreactive CD4 T cells
from a Th1 (IFN-
) to a Th2 (IL-4, IL-10) profile (13).
This possibility was tested by the introduction of a functionally
inactivated IL-4 gene into our Ea transgenic NOD stock.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD/Lt mice are maintained in a specific pathogen-free research colony at The Jackson Laboratory (Bar Harbor, ME). Currently, IDDM develops in 90% of female and 63% of male NOD/Lt mice by 1 yr of age. A stock of NOD mice made completely IDDM resistant by direct introduction of an H2-Ead transgene (formal designation NOD/Lt-TgN (H2-Ead)Ln5; here designated NOD-H2-E for simplicity) has been previously described (13). Derivation of an N10 backcross stock of NOD mice made IDDM resistant through the elimination of B lymphocytes by a functionally disrupted Igµ allele (official designation NOD.Igh6tm1Cgn; here designated NOD.Igµnull), and fixed to homozygosity for linkage markers delineating all previously identified Idd loci of NOD origin has also been previously described (20). These latter two stocks served as progenitors for a line of B lymphocyte-deficient NOD-H2-E mice (designated NOD-H2-E.Igµnull). NOD-H2-E and NOD.Igµnull mice were intercrossed. The resulting F1 hybrids were backcrossed to NOD.Igµnull mice to produce progeny homozygous for the Igµnull mutation and heterozygous for the H2-E transgene. Homozygous Igµnull segregants were identified by the absence of B lymphocytes among PBL using the previously described flow cytometric technique (20). Heterozygous H2-E transgene carriers were identified by the previously described Southern blot analysis of tail snip DNA (13). We had also previously produced (21, 22) an N9 backcross stock of NOD mice homozygous for both a functionally disrupted IL-4 gene and linkage markers delineating all known Idd loci of NOD origin (formal designation NOD.Il4tm1Cgn; here designated NOD.IL4null). The availability of this stock allowed for the generation of IL-4-deficient NOD-H2-E mice (designated NOD-H2-E.IL4null). F1 hybrids between NOD.IL4null and NOD-H2-E mice were backcrossed to the NOD.IL4null stock. Segregants homozygous for the IL4null allele were detected by the previously described PCR assay (21), while carriers of the H2-E transgene were identified by flow cytometry for positive staining of PBL with the FITC-conjugated monoclonal Ab 14-4-4S. All mice were allowed free access to food (National Institutes of Health 31A/6% fat diet; Ralston Purina, Richmond, IN) and acidified drinking water.
Assessment of diabetes development
IDDM development in the indicated mice was assessed by weekly
monitoring of glycosuric values with Ames Diastix (supplied by Bayer,
Diagnostics Division, Elkhart, IN). Values of 
3 were considered
indicative of IDDM onset.
Generation of mixed bone marrow/B lymphocyte chimeras
In some experiments, 4- to 6-wk-old female NOD mice were lethally irradiated (1200 rad from a 137Cs source) and reconstituted as previously described (23) with 5 x 106 T cell-depleted syngeneic bone marrow cells admixed with various numbers of T cell-depleted marrow cells from the indicated partner donor strains. Other experiments used female NOD.Igµnull mice reconstituted at 4–6 wk of age with 5 x 106 T cell depleted syngeneic marrow cells admixed with the indicated numbers of purified B lymphocytes from standard NOD and/or NOD-H2-E donors. Splenic B lymphocytes were purified using the previously described magnetic bead system (16). The purity of B lymphocytes isolated by this technique routinely exceeded 93%. Another experimental group consisted of NOD.Igµnull females reconstituted at 4–6 wk of age with 5 x 106 T cell-depleted NOD-H2-E.Igµnull marrow cells combined with 3 x 106 purified NOD B lymphocytes. Positive controls for all experimental groups consisted of NOD or NOD.Igµnull females reconstituted at 4–6 wk of age with 5 x 106 T cell-depleted NOD bone marrow cells. Control and experimental chimeras were monitored for IDDM development as described above for 21-wk postreconstitution. Upon developing IDDM or reaching the 21-wk postreconstitution end point, types and proportions of H2-E expressing APC in the various chimeras were determined by FACS analysis as described below. In addition, pancreases from chimeras remaining free of overt IDDM for the 21-wk postreconstitution period were histologically examined for insulitis development as described below.
Quantification of H2-E-expressing APC
Splenic leukocytes from the indicated chimeras were assessed by multicolor FACS analysis (FACScan; BD Biosciences, San Jose, CA) for proportions and types of APC expressing H2-E at the time of IDDM development or at 21 wk postreconstitution. Previous analyses found that maximal chimerization levels of all pertinent cell types were achieved at 4–6 wk postreconstitution and did not vary after this time. All analyses used the CellQuest 3.0 data reduction system (BD Biosciences). Total numbers of APC were defined by the presence of MHC class II expression. All APC in each type of chimera used in these studies expressed the NOD H2-Ag7 MHC class II gene product, which was detected with mAb AMS32.1 conjugated to a red fluorescent PE tag. The proportion of total APC that also expressed the H2-E MHC class II transgene product was detected by costaining splenic leukocytes with mAb 14-4-4S conjugated to a green fluorescent FITC tag. Separate aliquots of splenic leukocytes were assessed for costaining with the FITC-conjugated 14-4-4S Ab and the PE-conjugated RA3-6B2 Ab specific for the B220 cell surface marker to determine the proportion of B lymphocytes that expressed H2-E. The proportions of macrophages/DC that expressed H2-E were determined by costaining of splenic leukocytes with the FITC-conjugated 14-4-4S Ab and the PE-conjugated M1/70 Ab specific for the Mac-1 cell surface marker. Data are presented as the mean proportion ± SEM of total APC, B lymphocytes, or macrophages/DC that expressed H2-E in the indicated experimental group.
Histological analyses of insulitis development
The indicated chimeric mice remaining free of overt IDDM for
21-wk postreconstitution were assessed for insulitis development.
Pancreases were fixed in Bouin’s solution and sectioned at three
nonoverlapping levels. Granulated cells were stained with aldehyde
fuchsin, and leukocytes were stained with a H&E counterstain. Islets
(at least 20/mouse) were individually scored as follows: 0, no lesions;
1, peri-insular leukocytic aggregates, usually periductal infiltrates;
2, <25% islet destruction; 3, >25% islet destruction; and 4,
complete islet destruction. An insulitis score for each mouse was
obtained by dividing the total score for each pancreas by the number of
islets examined. Data are presented as the mean insulitis score
(MIS) ± SEM for the indicated experimental group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous transgenic studies in NOD mice indicated that a protocol that induces the expression of a dominantly protective MHC class II variant in APC might ultimately prove to be clinically useful in blocking IDDM development in otherwise susceptible individuals. However, a necessary prerequisite for such a protocol is to determine what subtype(s) of APC must express a particular protective MHC class II variant to most efficiently inhibit IDDM. We had previously found that a major component of IDDM resistance elicited by transgenic restoration of H2-E MHC class II expression in NOD mice entailed the induction of an extrathymic immunoregulatory process (13). B lymphocytes are rare in the thymus, but represent the most prevalent subtype of APC in the periphery. Thus, we hypothesized that the peripheral IDDM protective immunoregulatory process engendered by transgenic H2-E expression in NOD mice is mediated by B lymphocytes. We tested this possibility through use of our previously described chimeric system (16) that allowed NOD T cells to mature and function in an environment where transgenic H2-E expression is solely restricted to a selected proportion of B lymphocytes. These chimeras were generated by reconstituting lethally irradiated NOD.Igµnull recipients with syngeneic marrow admixed with various proportions of purified B lymphocytes from NOD and/or NOD-H2-E donors. This particular chimeric approach must be used because unmanipulated NOD.Igµnull mice are not tolerant of even standard NOD B lymphocytes and hence reject them upon direct infusion (16).
Table I
summarizes the mean
proportion of H2-E-positive and -negative B lymphocytes that
repopulated spleens of each type of chimera analyzed. Also depicted is
the proportion of total APC (defined as all MHC class II-positive
cells) in each type of chimera that coexpressed H2-E. Fig. 1 shows IDDM development as a function of
the percentage of H2-E-expressing splenic B lymphocytes that developed
in each group of mixed chimeras. As expected from a previous study
(16), IDDM developed over a 21-wk follow-up period in most
NOD.Igµnull females (65.0%, 15
of 23) reconstituted with syngeneic marrow and standard
H2-E-negative NOD B lymphocytes (Fig. 1). In contrast, over the same
period of time IDDM developed in 11.1% (one of nine) of
NOD.Igµnull females reconstituted with
syngeneic marrow and only NOD-H2-E B lymphocytes.
NOD.Igµnull mice that remained free of
overt IDDM following reconstitution with syngeneic marrow admixed only
with NOD-H2-E B lymphocytes were also characterized by
moderate levels of insulitis (MIS, 1.90 ± 0.49; n
= 8). The above chimeras expressed H2-E solely on all B lymphocytes
(80.8% of all APC). To address the minimal proportions of
H2-E-expressing B lymphocytes required to elicit IDDM resistance, we
also analyzed chimeras in which H2-E was expressed on 0–25, 25–50, or
>50% of B lymphocytes (Table I). This corresponded to mean
proportions of 13.6, 34.9, and 62.8% of total APC expressing H2-E.
Unlike the chimeras in which all B lymphocytes expressed H2-E, none of
the chimeras characterized by these lower levels of H2-E-expressing B
lymphocytes was significantly protected from IDDM (Fig. 1). Hence, NOD
B lymphocytes transgenically expressing H2-E MHC class II molecules can
inhibit IDDM development, but cannot do so when H-2-negative B
lymphocytes are also present. An important implication of these results
is that transgenic H2-E expression on all B lymphocytes is unlikely to
inhibit IDDM development in NOD mice by increasing the ability of these
APC to mediate a protective peripheral immunoregulatory mechanism.
Rather, when all NOD B lymphocytes express transgenic H2-E molecules,
they most likely lose some pathogenic function(s) normally necessary to
IDDM development.
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The preceding results indicated that H2-E-expressing B lymphocytes
cannot efficiently inhibit IDDM development in NOD mice. This suggested
that a significant level of protection might be achieved when H2-E was
exclusively expressed on macrophages/DC. However, if H2-E must be
simultaneously expressed on multiple types of APC to confer significant
protection from IDDM, then restricting expression of such class II
molecules solely to macrophages/DC would also prove to be an
ineffective means of inhibiting disease development. To distinguish
between these possibilities, we determined the extent of IDDM
development in chimeric NOD mice that expressed H2-E on all
macrophages/DC, but not B lymphocytes. These were generated by
reconstituting NOD.Igµnull female
recipients with NOD-H2-E.Igµnull bone
marrow admixed with purified standard NOD B lymphocytes. Controls
consisted of NOD.Igµnull female
recipients reconstituted with syngeneic marrow plus NOD B lymphocytes.
As expected, over a 21-wk follow-up period, IDDM developed in most
(71.4%, five of seven) of these control chimeras (Fig. 2). In contrast, over the same period of
time, IDDM developed in a significantly lower proportion (16.7%, 2 of
12) of the NOD.Igµnull females
reconstituted with NOD-H2-E.Igµnull bone
marrow plus NOD B lymphocytes. Furthermore, the
NOD.Igµnull females that remained free
of overt IDDM following reconstitution with
NOD-H2-E.Igµnull bone marrow plus NOD B
lymphocytes were characterized by significantly lower levels of
insulitis (MIS, 1.33 ± 0.36; n = 10) than the few
control chimeras that remained free of overt disease (MIS, 3.05 ±
0.94; n = 2). Hence, IDDM was inhibited in NOD mice
under conditions where transgenic H2-E molecules are expressed on all
macrophages/DC, but not any B lymphocytes.
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We hypothesized that if H2-E-expressing macrophages/DC inhibited
IDDM by inducing an active immunoregulatory mechanism, then they would
only need to comprise some minimal threshold proportion of all APC to
manifest their protective effect. To initially test this possibility,
we compared the extent of IDDM development in standard NOD female mice
reconstituted with syngeneic marrow alone or admixed with an equal
number of marrow cells from NOD-H2-E donors. As expected,
about 50% of all APC in NOD females reconstituted with a 1/1 mixture
of NOD and NOD-H2-E marrow expressed H2-E (Table II). It is important to note that unlike
the mice depicted in Fig. 1
in which the only H2-E-expressing APC were
B lymphocytes, in this experiment the 50% of H2-E-expressing APC also
included macrophages/DC. Fig. 3 shows
that over the 21-wk observation period, IDDM developed in a
significantly smaller proportion of the chimeras reconstituted with a
1/1 mixture of NOD and NOD-H2-E marrow (33.3%, 8 of 24),
than NOD marrow alone (91.7%, 11 of 12). Insulitis levels in chimeras
that did not develop overt IDDM following reconstitution with a 1/1
mixture of NOD and NOD-H2-E marrow (MIS, 1.43 ± 0.3;
n = 14) were also much lower than that in the single
control chimera that failed to develop overt disease (IS, 3.9)
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, H2-E expression solely on 
50% of B lymphocytes
did not inhibit IDDM development. Hence, the ability of H2-E to also be
expressed on macrophages/DC most likely accounted for disease
protection in the chimeras depicted in Fig. 3 that were reconstituted
with a 1/1 mixture of NOD and NOD-H2-E marrow. To test this
hypothesis, we assessed IDDM development in chimeras where transgenic
H2-E molecules were only expressed on a fraction of macrophages/DC, but
not on B lymphocytes. This was done by reconstituting NOD female
recipients with syngeneic marrow admixed with an equal number of marrow
cells from NOD-H2-E.Igµnull donors. Such
an approach generated chimeras that could be stratified on the basis of
H2-E-expressing macrophages/DC comprising either more or less than 5%
of the total APC (Table II). As shown in Fig. 3, the only chimeras that
developed IDDM at a rate significantly less (42.9%, three of seven)
than controls reconstituted with NOD marrow alone (91.7%), were those
in which H2-E-expressing macrophages/DC comprised >5% of the total
APC. In these IDDM-resistant chimeras, the mean proportion of
H2-E-expressing macrophages/DC among total MHC class II-positive APC
was 7.3 ± 0.5% (Table II). This value represents a little over
half of the total macrophages/DC. Such a finding, combined with the
fact that chimeras reconstituted with a 1/1 mixture of NOD and
NOD-H2-E marrow are also IDDM resistant, indicates that
H2-E-mediated disease protection requires that these MHC class II
molecules be expressed on about 50% of macrophages/DC, which
represents about 5% of all APC. Production of the Th2 cytokine IL-4 is not required for the induction of IDDM resistance in NOD-H2-E mice
A number of Ag-specific and nonspecific immunostimulatory
protocols that inhibit IDDM development in NOD mice are associated with
a shift in the cytokines produced by islet-infiltrating T cells from a
Th1 (primarily IFN-) to a Th2 (primarily IL-4) profile (reviewed in
Refs. 1, 24 , and 25). We had previously found
that following priming and restimulation with the 65-kDa variant of the
candidate cell autoantigen glutamic acid decarboxylase, T cells
from the NOD-H2-E transgenic stock produced significantly
higher levels of the Th2 cytokine IL-4 than similar T cells from
standard NOD mice (13). Thus, we hypothesized that
H2-E-mediated IDDM inhibition might result from an increased ability of
cell autoreactive T cells to produce the potentially protective Th2
cytokine IL-4. We subsequently developed a fully IDDM-susceptible NOD
congenic stock genetically deficient in IL-4 (21). The
availability of this stock allowed us to produce through intercrossing,
NOD-H2-E transgenic mice with either an intact or a
genetically disrupted IL-4 gene. As expected, by 30 wk of age, IDDM had
developed in a high proportion (73.7%, 14 of 19) of female IL-4
intact, H2-E-negative, NOD control segregants (Fig. 4). Also, as expected based on previously
published results (22), over the same period of time IDDM
developed in a high proportion (68.8%, 11 of 16) of IL-4-deficient
H2-E-negative congenic females. However, transgenic H2-E expression
remained capable of completely suppressing IDDM development in
IL-4-deficient females (0%, 0 of 14). Hence, the inhibition of IDDM
development in NOD mice by transgenic restoration of H2-E MHC class II
expression is not dependent upon the induction of IL-4 production by
cell autoreactive T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5% of the total MHC class
II-positive APC. Significantly, the extent of IDDM protection observed
when H2-E was solely expressed on approximately half of the
macrophages/DC was the same as that obtained when H2-E was expressed on
half of all APC. This indicates that transgenic restoration of H2-E
expression on half of the macrophages/DC and B lymphocytes does not
result in a synergistic enhancement of IDDM resistance. Collectively,
these results demonstrate that transgenic restoration of H2-E
expression on B lymphocytes or macrophages/DC can inhibit IDDM
development in NOD mice, but there are differences in the proportions
of these APC subtypes that must express H2-E to elicit protective
effects. Furthermore, based on their ability to inhibit disease at
quantitatively lower levels than B lymphocytes, macrophages/DC appear
to represent the most efficient subtype of APC for mediating
H2-E-induced IDDM protection.
There are several mechanistic implications of the finding that
different percentages of B lymphocytes vs macrophages/DC must express
H2-E to exert IDDM protective effects in NOD mice. The first of these
is that complete IDDM resistance in the NOD-H2-E stock (all
APC express H2-E molecules) should additively result from the partially
protective effects that are engendered when H2-E is expressed on all B
lymphocytes and at least 50% of macrophages/DC. Furthermore, the
finding that the majority, if not all, B lymphocytes must express H2-E
to inhibit IDDM development indicates such protection does not result
from this APC subset acquiring an ability to actively mediate an
immunoregulatory mechanism(s). Rather, the expression of H2-E on most
or all B lymphocytes most likely results in their loss of some
pathogenic function(s) normally necessary to IDDM development. Such
lost pathogenic functions could include those that allow B lymphocytes
to serve as a preferential subpopulation of APC for activating
autoreactive CD4 T cell responses against certain cell autoantigens
(16, 17). In contrast, the finding that H2-E only needs to
be expressed on a portion of macrophages/DC to inhibit IDDM development
in NOD mice suggests that such protection results from these APC
acquiring the ability to activate a normally absent immunoregulatory
function(s).
The question remains as to what sort of active IDDM protective immunoregulatory activities are gained by NOD macrophages/DC that transgenically express H2-E molecules. Macrophages/DC represent the most prevalent type of APC in the thymus. Hence, such cells are likely to mediate the previously reported intrathymic deletion of a highly diabetogenic CD4 T cell clonotype in H2-E-expressing NOD mice (27). However, H2-E expression does not result in the clonal deletion of a broad range of diabetogenic CD4 T cells. This was demonstrated by the finding that purified T cells from NOD-H2-E mice (liberated from the influence of H2-E-expressing APC) could adoptively transfer IDDM to lymphocyte-deficient NOD-scid recipients (13). These previous results also indicate that NOD diabetogenic T cells remain functionally suppressed, but are not anergized, as long as they remain in the presence of H2-E-expressing APC. Our current findings indicate that macrophages/DC represent the subtype of H2-E-expressing APC that maintain the peripheral functional suppression of diabetogenic NOD T cells.
We had previously obtained evidence that H2-E-expressing APC might
functionally suppress cell autoreactive CD4 T cells in NOD mice by
enhancing their production of the Th2 cytokine IL-4, which has been
proposed to dampen the pathogenic potential of such effectors (1, 13, 24, 25). This hypothesis was rejected in the current study,
because transgenic restoration of H2-E expression remained capable of
completely inhibiting IDDM in NOD mice made genetically deficient in
IL-4. It has also been proposed that through a mechanism termed
determinant capture, transgenic H2-E molecules can inhibit IDDM
development in NOD mice by binding pancreatic cell peptides that
are normally presented to autoreactive CD4 T cells by
H2-Ag7 class II molecules, hence preventing the
activation of such effectors (28). Partially arguing
against the determinant capture mechanism is a report that none of 80
peptides derived from the candidate cell autoantigen 65-kDa heat
shock protein could strongly bind to both H2-Ag7
and transgenic H2-E MHC class II molecules (29). We had
also previously found that transgenic H2-E expression might inhibit
IDDM development in NOD mice by inducing a reciprocal decrease in the
expression of endogenous H2-Ag7 MHC class II
molecules on APC (13). However, it is difficult to
envision that such a decrease in H2-Ag7
expression could increase the ability of APC to mediate an active
immunoregulatory function such as that exerted by macrophages/DC from
NOD-H2-E mice. In contrast, the triggering of a reciprocal
decrease in H2-Ag7 expression on most or all B
lymphocytes in NOD-H2-E mice could account for this subset
of APC losing a pathogenic function(s) normally necessary for IDDM
development, such as their preferential ability to present certain
cell Ags to autoreactive CD4 T cells. Thus, while our current studies
clearly demonstrate that IDDM resistance in NOD-H2-E mice is
mediated through different effects by both B lymphocytes and
macrophages/DC, the actual protective mechanisms exerted by these APC
subsets remain an open question.
Our current results also illustrate an important obstacle that will
have to be overcome before it will be possible to use APC made to
express a single dominantly protective MHC class II gene product for
blocking IDDM development in a clinical setting. This obstacle is that
the clinical induction of IDDM resistance may require the expression of
the appropriate class II variant in a very high proportion of a
particular subtype of APC. In the case of H2-E in NOD mice, protection
requires expression of this class II variant in at least half of all
macrophages/DC and/or the vast majority of B lymphocytes. It would
probably be difficult to maintain this level of protective APC by their
direct infusion into IDDM-susceptible individuals. Instead, the most
effective way of maintaining the necessary level of protective APC in
such individuals would be to reconstitute them with essentially pure
populations of hemopoietic stem cells that had been transfected with
the appropriate MHC class II gene construct. Hemopoietic stem cells are
extremely rare within bone marrow (1 in 105
cells), and while much progress has been made, methodologies allowing
for their purification have remained elusive (reviewed in Refs.
30 and 31). Thus, major advances will have to
be made in hemopoietic stem cell purification technologies to
subsequently develop a clinical protocol for inhibiting IDDM through
the use of APC expressing dominantly protective MHC class II gene
products.
In conclusion, we have found that the induction of IDDM resistance in
NOD mice by transgenic restoration of H2-E MHC class II expression is
mediated by multiple subtypes of APC through different, but additive,
mechanisms. Only when H2-E is expressed on most or all B lymphocytes
does this APC subset lose some pathogenic function(s) normally
necessary for IDDM development. In contrast, H2-E expression on some
critical threshold level of macrophages/DC (50%) in NOD mice
results in the induction of an active immunoregulatory process that
functionally suppresses, but does not anergize, autoreactive
diabetogenic T cells. Hence, macrophages/DC would appear to be the APC
subset that represents the most rational target for therapies that are
designed to inhibit IDDM development through the expression of
dominantly protective MHC class II alleles.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David V. Serreze, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609. E-mail address: dvs{at}jax.org
3 Abbreviations used in this paper: IDDM, type 1 diabetes, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; MIS, mean insulitis score.
Received for publication February 27, 2001. Accepted for publication June 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain or normal I-E -chain. Nature 345:727.[Medline]
cells of nonobese diabetic mice. J. Immunol. 163:743.-transgenic nonobese diabetic mice from autoimmune diabetes. J. Immunol. 162:6630.This article has been cited by other articles:
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  Y.-G. Chen, P. A. Silveira, M. A. Osborne, H. D. Chapman, and D. V. Serreze Cellular Expression Requirements for Inhibition of Type 1 Diabetes by a Dominantly Protective Major Histocompatibility Complex Haplotype Diabetes, February 1, 2007; 56(2): 424 - 430. [Abstract] [Full Text] [PDF]
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 D. V. Serreze, M. A. Osborne, Y.-G. Chen, H. D. Chapman, T. Pearson, M. A. Brehm, and D. L. Greiner Partial versus Full Allogeneic Hemopoietic Chimerization Is a Preferential Means to Inhibit Type 1 Diabetes as the Latter Induces Generalized Immunosuppression J. Immunol., November 15, 2006; 177(10): 6675 - 6684. [Abstract] [Full Text] [PDF]
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P. A. Silveira, H. D. Chapman, J. Stolp, E. Johnson, S. L. Cox, K. Hunter, L. S. Wicker, and D. V. Serreze Genes within the Idd5 and Idd9/11 Diabetes Susceptibility Loci Affect the Pathogenic Activity of B Cells in Nonobese Diabetic Mice J. Immunol., November 15, 2006; 177(10): 7033 - 7041. [Abstract] [Full Text] [PDF]
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A. Ueno, S. Cho, L. Cheng, Z. Wang, B. Wang, and Y. Yang Diabetes Resistance/Susceptibility in T Cells of Nonobese Diabetic Mice Conferred by MHC and MHC-Linked Genes J. Immunol., October 15, 2005; 175(8): 5240 - 5247. [Abstract] [Full Text] [PDF]
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D. V. Serreze, T. M. Holl, M. P. Marron, R. T. Graser, E. A. Johnson, C. Choisy-Rossi, R. M. Slattery, S. M. Lieberman, and T. P. DiLorenzo MHC Class II Molecules Play a Role in the Selection of Autoreactive Class I-Restricted CD8 T Cells That Are Essential Contributors to Type 1 Diabetes Development in Nonobese Diabetic Mice J. Immunol., January 15, 2004; 172(2): 871 - 879. [Abstract] [Full Text] [PDF]
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M. A. Pierce, H. D. Chapman, C. M. Post, A. Svetlanov, S. Efrat, M. Horwitz, and D. V. Serreze Adenovirus Early Region 3 Antiapoptotic 10.4K, 14.5K, and 14.7K Genes Decrease the Incidence of Autoimmune Diabetes in NOD Mice Diabetes, May 1, 2003; 52(5): 1119 - 1127. [Abstract] [Full Text] [PDF]
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;
n = 23) or NOD-H2-E (
;
n = 9) donors. Other
NOD.Igµnull female mice were
reconstituted with syngeneic marrow together with three different
mixtures of NOD and NOD-H2-E B lymphocytes (group 1,
5 x 106 of each; group 2, 2 x
106:6 x 106; group 3, 1 x
106:10 x 106). This respectively produced
chimeras in which 0–25% (
; n = 21), 25–50%
(
; n = 14), or >50% (
;
n = 6) of the B lymphocytes expressed H2-E. All
chimeras were monitored for IDDM development for 21-wk
postreconstitution. *, IDDM development significantly less
(p < 0.05, Kaplan-Meier life table analysis) than
in NOD.Igµnull controls
reconstituted with syngeneic marrow and NOD B lymphocytes.
